In humans and other mammals, fibronectin is a dimeric glycoprotein found at high concentrations in plasma and other body fluids. D. Mosher et al., Fibronectin, New York, Academic Press Inc. (1989); R; Hynes, Integrins: Versatility, modulation, and signaling in cell adhesion, Cell 69:11-26 (1982). The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
Soluble protomeric fibronectin is found at high concentrations in plasma and other body fluids and is synthesized and secreted in vitro by many cell types. Insoluble fibronectin consists of high molecular weight disulfide-stabilized multimers of fibronectin and is found in connective tissue, basement membranes and extracellular matrices. It mediates many important biological functions. Some of these are cellular migration during embryogenesis, wound healing and tumor metastasis. J. Labat-Robert et al., Comparative distribution patterns of Type I and III collagen and fibronectin in human arteriosclerotic aorta, Pathol. Biol. (Paris) 33, 261-265. (1985); R. Colvin, Fibronectin in wound healing. In Fibronectin. D. F. Mosher, ed. (New York: Academic Press) pp. 213-254 (1989); R. Hynes, Integrins: Versatility, modulation and signaling in cell adhesion, Cell 69, 11-26 (1992).
Insolubilization of cellular fibronectin is initiated by the reversible binding of soluble fibronectin to cell surfaces. Bound fibronectin is subsequently deposited into high-molecular-weight fibronectin multimers. Thus, the process of fibronectin matrix assembly is a stepwise process.
In the first step, soluble protomeric fibronectin reversibly binds to a putative cell surface matrix assembly receptor via an amino-terminal 70 KDa region of fibronectin. Once fibronectin has bound to the cell surface, additional fibronectin domains may participate in matrix assembly. In particular, fibronectin-fibronectin interactions may serve to further stabilize fibronectin binding and assure proper alignment of the incoming fibronectin. Finally, the fibronectin is stabilized within the fibril. Fibronectin matrix assembly (and thus wound healing, tumor metastasis, etc.) depends upon the fibronectin first efficiently and effectively binding to cell surfaces.
Certain serum components have been reported to improve fibronectin binding to a fibroblastic monolayer culture. This correlates with the finding that transformed hamster cells grown in 5% serum exhibit increased fibronectin deposition compared to cells grown in 0.3% serum. A serum component, transforming growth factor .beta., has been found to increase cell surface binding and assembly of exogenous plasma fibronectin by fibroblasts. See B. Allen-Hoffmann, et al., Transforming Growth Factor .beta. Increases Cell surface Binding and Assembly of Exogenous (Plasma) Fibronectin by Normal Human Fibroblasts, MCB 8, 4234-4242 (1988). Recently, our lab identified lipoproteins as additional enhancers of fibronectin binding to adherent cells. W. Checovich, et al., Lipoproteins Enhance Fibronectin Binding to Adherent Cells. Arterioscl. and Thromb. 12:1122-1130 (1992).
However, multi-component materials (e.g. serum; lipoproteins) cannot be used efficiently for certain applications (given that they have many components in them unrelated to fibronectin binding), and certain serum components have undesirable properties.
In unrelated research, there have been various studies of lysophosphatidic acids ("LPA"). As shown in FIG. 1 (1oleoyl, lysophosphatidic acid), a lysophosphatidic acid is an acid in which only one of the hydroxyl groups of the glycerol is esterified. Historically, the prefix lysoderives from the fact that the acids are often good detergents (and as such often can "lyse" cells). However, more generically, they are phosphatidic acids where the carbon is not esterified and the "3" carbon is bound to the O--PO.sub.3 H.sub.2 group, or in the case of the salt one or more hydrogens are replaced (e.g. with Na.sup.+). The "1" carbon will contain an acyl ester (in nature typically C.sub.10 -C.sub.30 ; most often C.sub.14 -C.sub.24) of fatty acids. Preferred LPAs are 1-acyl-SN-glycerol-3 phosphates.
Extracellular LPA is known to evoke diverse physiological responses, such as platelet aggregation, smooth muscle contraction, and fibroblast proliferation. Furthermore, LPA is known to function as a Ca.sup.++ -mobilizing agonist for a great variety of cell types. It has also been shown that exogenous LPA stimulates phospholipid hydrolysis via activation of phospholipase C or D, with subsequent Ca.sup.++ mobilization and stimulation of protein kinase C, and that LPA can inhibit adenylate cyclase in a G.sub.i -protein dependent manner.
Also, it has been found that LPA-induces shape changes in nerve cells and that LPA stimulates mitogen-activated protein (MAP) kinase by a G-protein-coupled process.
In A. Ridley, et al., The Small GTP-Binding Protein rho Regulates the Assembly of Focal Adhesions and Action Stress Fibers in Response to Growth Factors, Cell 70, 389-399 (1992), it was reported that LPA also is responsible for the ability of serum to cause actin stress fiber formation at focal adhesions inside cells.
LPA has also been shown to affect the binding of fibrinogen (as distinguished from fibronectin) to isolated platelet glycoprotein IIb-IIIa. S. Smyth, et al., Fibrinogen binding to purified platelet glycoprotein IIb-IIIa is modulated by lipids, J. Biol. Chem. 267: 155568-155577 (1992).
However, prior to our work, LPA had not been suggested to promote fibronectin binding on the external surfaces of cells.